Compounds modulating parathyroid hormone (PTH) secretion

The control of parathyroid hormone (PTH) secretion is strictly regulated by the parathyroid Ca receptor (CaR). Calcimimetics and calcilytics selectively act on the parathyroid CaR to inhibit and enhance PTH secretion, respectively. According to the recent pharmacological two-state model, calcimimetics act on the CaR as allosteric agonists to stabilize an active conformation of CaR. Conversely, calcilytics act on the CaR as allosteric inverse agonists to stabilize an inactive conformation of CaR. These compounds that can alter circulating levels of PTH and bone turnover might provide novel treatments for adynamic bone disease in patients with chronic renal failure.     

Mitogen-activated protein kinase cascade in human normal and tumoral parathyroid cells

The calcium-sensing receptor (CaR) activation has recently been shown to modulate the ERK1 and ERK2 cascade in different cell lines. The present study investigated this pathway in human normal and tumoral parathyroid cells. In cells from normal parathyroids and almost all hyperplasia increasing extracellular calcium concentrations (Ca(o)(2+)) induced a significant activation of ERK1 and -2, the percent stimulation over basal activity (at 0.5 mM Ca(o)(2+)) being 545 +/- 140 and 800 +/- 205 in normal cells and 290 +/- 71 and 350 +/- 73 in hyperplasia at 1 and 2 mM Ca(o)(2+), respectively. This effect was mediated by CaR because it was mimicked by the receptor agonist gadolinium and neomycin. Basal and Ca(o)(2+)-stimulated ERK1 and -2 activity was nearly abolished by the PKC inhibitor calphostin C, and PKA changes did not affect ERK1 and -2 activity. PI3K blockade by wortmannin, known to prevent G protein betagamma subunit effect on ERK1 and -2, induced a 30% reduction of the Ca(o)(2+)-stimulated ERK1 and -2 activity. Adenomatous cells showed high PKC-dependent ERK1 and -2 activity in resting conditions that was unresponsive to high Ca(o)(2+). A role of MAPK on PTH secretion was suggested by the finding that PD98059, a specific MEK inhibitor, abolished the inhibitory effect of 1.5 mM Ca(o)(2+) on PTH release from normal parathyroid cells. In conclusion, these data first demonstrate that CaR activation, through the PKC pathway and, to a lesser extent, PI3K, increases ERK1 and -2 activity in normal parathyroid cells and this cascade seems to be involved in the modulation of PTH secretion by Ca(o)(2+). Interestingly, this signaling pathway is disrupted in parathyroid tumors.     

Parathyroid expression of calcium-sensing receptor protein and in vivo parathyroid hormone-Ca(2+) set-point in patients with primary hyperparathyroidism

A reduced expression of calcium-sensing receptor (CaR) messenger ribonucleic acid and protein accompanied by abnormalities in parathyroid cell proliferation and PTH secretion are present in primary hyperparathyroidism. We studied the expression of CaR protein by immunohistochemistry in 36 sporadic parathyroid adenomas and investigated the relationship between CaR expression and several preoperative clinical parameters, including the set-point of Ca(2+)-regulated PTH secretion (measured in vivo). The adenomas were classified in 4 categories according to the intensity of immunohistochemical staining: 5 (14%) showed a CaR staining intensity similar to that of normal parathyroid ( ), 10 (27%) showed moderate staining (++), 16 (45%) showed weak staining (+), and 5 (14%) were negative (-). The intensity of CaR staining was not related to preoperative serum Ca(2+), PTH levels or adenoma volume. Twenty-nine patients underwent preoperatively the calcium infusion test to evaluate the PTH-Ca(2+) set-point. Individual values of PTH-Ca(2+) set-point ranged from 1.38-1.93 mmol/L and were significantly correlated with basal Ca(2+) levels (r = 0.96; P: = 0. 0001) and adenoma volume (r = 0.5; P: = 0.01). The mean PTH-Ca(2+) set-point values were significantly different in the 4 groups of patients classified according to immunohistochemical staining intensity of their adenoma (P: = 0.025; F = 3.78); the mean PTH-Ca(2+) set-point was significantly higher in the groups classified as negative than in those classified as weak or moderate. No correlation was observed between the PTH-Ca(2+) set-point and basal PTH levels or between the percent maximal PTH inhibition and adenoma volume and basal PTH or Ca(2+) levels. In summary, our data suggest that there is a relationship between apparent CaR protein expression and PTH-Ca(2+) set-point abnormality, suggesting that a reduced receptor content might have an important role in the pathogenesis of primary hyperparathyroidism.     

Parathyroid gland calcium receptor mRNA levels are unaffected by chronic renal insufficiency or low dietary calcium in rats

Extracellular ionized calcium (Ca(2+)) is the primary physiological regulator of parathyroid hormone (PTH) secretion and the G protein-coupled receptor (CaR) that mediates this response has been cloned from bovine and human parathyroid glands. The Ca(2+) set-point for the regulation of PTH secretion is right-shifted in primary hyperparathyroidism (1°HPT), but whether there is a similar shift in 2°HPT is unclear. Additionally, the molecular defects associated with such changes in the set-point remain uncharacterized. These experiments were designed to determine (1) if changes in set-point occur in rats with 2°HPT induced by chronic renal insufficiency (CRI) or dietary Ca deficiency, and (2) whether any changes in set-point are mirrored by changes in steady-state mRNA levels for the parathyroid CaR. CaR mRNA levels were quantified in pairs of glands from individual rats using a solution hybridization assay. Blood urea nitrogen and PTH levels were ～ 4-fold higher in rats with CRI induced by 5/6 nephrectomy 7 weeks earlier. Rats with CRI were also significantly hypocalcemic and hyperphosphatemic. The setpoint was unchanged in CRI rats and CaR mRNA levels were also unaffected. Normal rats fed a 0.02% Ca diet for 6 weeks were markedly hypocalcemic, and had 10- and 15-fold increases in plasma PTH and 1,25-dihydroxyvitamin D(3) levels, respectively. Technical problems prevented assessment of the set-point in these animals, but parathyroid gland CaR mRNA levels were identical in both dietary groups. Thus, neither alterations in mRNA levels for the CaR nor changes in the set-point play demonstrable roles in the pathogenesis of 2°HPT in these models.     

Relationship between parathyroid calcium-sensing receptor expression and potency of the calcimimetic, cinacalcet, in suppressing parathyroid hormone secretion in an in vivo murine model of primary hyperparathyroidism

Cinacalcet HCl, an allosteric modulator of the calcium-sensing receptor (CaR), has recently been approved for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease on dialysis, due to its suppressive effect on parathyroid hormone (PTH) secretion. Although cinacalcet's effects in patients with primary and secondary hyperparathyroidism have been reported, the crucial relationship between the effect of calcimimetics and CaR expression on the parathyroid glands requires better understanding. To investigate its suppressive effect on PTH secretion in primary hyperparathyroidism, in which hypercalcemia may already have stimulated considerable CaR activity, we investigated the effect of cinacalcet HCl on PTH-cyclin D1 transgenic mice (PC2 mice), a model of primary hyperparathyroidism with hypo-expression of CaR on their parathyroid glands. A single administration of 30 mg/kg body weight (BW) of cinacalcet HCl significantly suppressed serum calcium (Ca) levels 2 h after administration in 65- to 85-week-old PC2 mice with chronic biochemical hyperparathyroidism. The percentage reduction in serum PTH was significantly correlated with CaR hypo-expression in the parathyroid glands. In older PC2 mice (93-99 weeks old) with advanced hyperparathyroidism, serum Ca and PTH levels were not suppressed by 30 mg cinacalcet HCl/kg. However, serum Ca and PTH levels were significantly suppressed by 100 mg/kg of cinacalcet HCl, suggesting that higher doses of this compound could overcome severe hyperparathyroidism. To conclude, cinacalcet HCl demonstrated potency in a murine model of primary hyperparathyroidism in spite of any presumed endogenous CaR activation by hypercalcemia and hypo-expression of CaR in the parathyroid glands.     

The influence of the progression of secondary hyperparathyroidism on the set point of the parathyroid hormone-calcium curve

The influence of secondary hyperparathyroidism (2 HPT) on the set point of the parathyroid hormone (PTH)-Ca(2+) curve is controversial. In vitro experiments have shown an increase in the set point. However, clinical studies with hemodialysis patients have provided a variety of results (increases, decreases and no changes in the set point have been reported). The present study was designed to investigate the influence of the progression of 2 HPT on the set point of the PTH-Ca(2+) curve. The PTH-Ca(2+) curve and the expression of parathyroid calcium receptor (CaR mRNA) and vitamin D receptor (VDR mRNA) have been studied in normal rabbits (group I, n=9) and in nephrectomized rabbits (group II, n=18) at two stages after inducing 2 HPT: 2-3 weeks (group IIA) and 5-6 weeks (group IIB). In group I, the set point of the PTH-Ca(2+) curve was 1.63+/-0.03 mM. A progressive hypocalcemia was detected during the evolution of 2 HPT (groups IIA and IIB). Rabbits from group IIA had a significant (P<0.001) decrease in the set point to values of 1.45+/-0.02 mM. However, the set point increased significantly in group IIB (P<0.001) to 1.56+/-0.03 mM. CaR mRNA was similarly decreased in groups IIA (39+/-12%) and IIB (48+/-7%). No changes were detected in VDR mRNA. In conclusion, a reduction in the set point of the PTH-Ca(2+) curve in response to decreased extracellular Ca(2+) was detected in the early phases of 2 HPT. However, with the progression of 2 HPT the set point tended to increase even though extracellular Ca(2+) was markedly decreased. The increase in the set point in the course of 2 HPT seems to be a complex process that cannot be fully explained by changes in parathyroid CaR mRNA or VDR mRNA.     

Phospholipase-A2 action and arachidonic acid metabolism in calcium-mediated parathyroid hormone secretion.

The involvement of arachidonic acid (AA) and its metabolites in the control of PTH secretion by porcine parathyroid cells was investigated. Increasing the extracellular calcium concentration from 0.5 to 2 mM increased free [3H]AA release and decreased PTH secretion from labeled parathyroid cells as a function of time (1-30 min). Free [3H]AA in the medium was significantly increased (+153 +/- 6%) after 5 min, while PTH secretion was significantly decreased (-75 +/- 7%) only after 15 min, suggesting a link between the two. [3H]AA release was associated with a decrease in [3H]AA incorporated into phosphatidylinositol, phosphatidic acid, and phosphatidylcholine, suggesting that these phospholipids are the major source of AA. Exogenous phospholipase-A2 (PL-A2; 1-500 mU/ml) and AA (5-40 microM) inhibited PTH secretion in a dose-dependent manner. PTH secretion inhibited by 2 mM Ca2+ was restored by two PL-A2 inhibitors, indomethacin (30 microM) and mepacrine (50 microM). The cyclooxygenase pathway inhibitor ibuprofen (20 microM) did not restore PTH secretion of affect high Ca(2+)-, AA-, or PL-A2-inhibited PTH secretion. Two inhibitors of the lipoxygenase pathway (LO), phenidone (1 microM) and baicalein (0.1 microM), a relatively selective 12-LO inhibitor, blunted high Ca(2+)-induced inhibition of PTH secretion (+101 +/- 10% and +105 +/- 6%, respectively), but nordihydroguaiaretic acid, which inhibits the 5-LO pathway, did not restore PTH secretion inhibited by high Ca2+, AA, or PL-A2. These results suggested that AA and agents that cause its liberation inhibit PTH secretion. AA may act via the 12-LO, but not via the 5-LO or cyclooxygenase, pathway. Thus, 12-LO products may be second messengers in parathyroid cells.     

Fluoride stimulates the accumulation of inositol phosphates, increases intracellular free calcium, and inhibits parathyroid hormone release in dispersed bovine parathyroid cells

The stimulation of polyphosphoinositide (PPI) turnover is associated with cellular activation and hormone secretion in numerous systems. GTP-binding proteins appear to couple receptors to phospholipase-C-mediated PPI breakdown. We assessed the effects of fluoride, an activator of GTP-binding proteins, on inositol phosphate accumulation, intracellular free Ca2+ [(Ca2+)i], cAMP content, and PTH release in dispersed bovine parathyroid cells. Sodium fluoride (5-30 mM) produced marked dose-dependent increases in inositol phosphates. With anion exchange HPLC, we confirmed that 30 mM fluoride stimulated a rapid increase in 1,4,5-inositol trisphosphate, a potent Ca2+-mobilizing compound. Using the Ca2+-sensitive probe fura-2, we determined that 30 mM fluoride increased [Ca2+]i from 339 +/- 9 to 650 +/- 39 nM (n = 8) within 30-60 sec at 1 mM extracellular Ca2+. After the depletion of extracellular Ca2+ by the addition of 1 mM EGTA, 30 mM fluoride increased [Ca2+]i 45 +/- 9% (n = 4), indicating that fluoride can mobilize intracellular Ca2+ stores. Fluoride (1-30 mM) also inhibited PTH release in dose-dependent fashion. Fluoride (30 mM) produced 72.8 +/- 4.2% suppression of maximal low Ca2+-stimulated PTH release comparable to the 83.7 +/- 3.7% inhibition by 2.0 mM extracellular Ca2+. Since changes in both [Ca2+]i and cAMP regulate PTH release, we measured the effect of fluoride on intracellular cAMP. Fluoride did not detectably change basal cAMP content, but it reduced forskolin-stimulated increases in cAMP. We conclude that fluoride may activate at least two GTP-dependent processes in parathyroid cells, resulting in PPI breakdown and cAMP accumulation. While both may contribute to the fluoride-induced suppression of PTH release, our findings suggest that the stimulation of PPI turnover leads to inhibition of PTH secretion.     

Relationship between parathyroid hormone secretion and cytosolic calcium concentration in dispersed bovine parathyroid cells.

The parathyroid cell is unusual among exocytotic systems in that low extracellular Ca2+ concentrations stimulate, while high Ca2+ concentrations inhibit, parathyroid hormone (PTH) release, suggesting that this cell might have unique secretory mechanisms. In the present studies, we used the Ca2+-sensitive fluorescent dye QUIN -2 to examine the relationship between cytosolic Ca2+ concentration and PTH release in dispersed bovine parathyroid cells. The secretagogue dopamine, which enhances PTH release 2- to 3-fold in association with 20- to 30-fold increases in cellular cAMP, had no effect on the cytosolic Ca2+ level (261 +/- 28 vs. 236 +/- 22 nM for control cells at 1 mM extracellular Ca2+; P greater than 0.05). Dibutyryl-cAMP, which produces a comparable stimulation of PTH release, likewise did not modify the level of cytosolic Ca2+. Removal of extracellular Ca2+ produced a further decrease of the cytosolic Ca2+ to 82 +/- 10 nM. However, PTH secretion persisted at a near maximal rate despite this decrease of extracellular and cytosolic Ca2+ and was 95 +/- 2.5% of the rate of hormonal release at 0.5 mM extracellular Ca2+. In contrast, addition of the divalent cation ionophore ionomycin to parathyroid cells at 1.0 mM extracellular Ca2+ inhibited PTH secretion in association with an increase in cytosolic Ca2+ from 230 +/- 13 nM to 570 +/- 50 nM. Moreover, the magnitude of the ionomycin-induced reduction in PTH secretion (64 +/- 4% relative to the secretory rate at 0.5 mM Ca2+) was equivalent to the inhibition of PTH release caused by 1.5 mM extracellular Ca2+ (64 +/- 6%), which increased the cytosolic Ca2+ to similar levels (450 +/- 48 nM). Thus, the parathyroid cell differs from secretory cells thought to operate by stimulus-secretion coupling in the following ways: changes in PTH release can occur without detectable alterations in the cytosolic Ca2+ concentration, maximal rates of PTH secretion occur at cytosolic Ca2+ concentrations that fail to support exocytosis in other cell types, and increases in the cytosolic Ca2+ concentration due to ionomycin inhibit rather than stimulate PTH release. Therefore, the control of PTH secretion by Ca2+ and other secretagogues may involve previously undefined mechanisms whereby hormonal release is relatively independent of the cytosolic Ca2+ at low levels of this parameter and is inversely related to cytosolic Ca2+ at higher levels of intracellular Ca2+.     

Drugs inhibiting parathyroid hormone (PTH) secretion by control of the calcium receptor (calcimimetics)--effect on the set point of calcium-regulated PTH secretion

Calcimimetics are positive allosteric modulators that activate the parathyroid calcium receptor (CaR) and thereby immediately suppress parathyroid hormone (PTH) secretion. Preclinical studies have demonstrated that calcimimetics inhibit PTH secretion and parathyroid gland hyperplasia and ameliorates bone qualities in rats with chronic renal insufficiency. Clinical trials with cinacalcet hydrochloride, a calcimimetic compound, have shown that calcimimetics possess lowering effects not only on serum PTH levels but also on serum phosphorus levels in dialysis patients with secondary hyperparathyroidism (2HPT). Thus, calcimimetics have considerable potential as an innovative medical approach to manage 2HPT. In this review, the similarities are extrapolated between the pharmacological effect of calcimimetics on the set point of Ca-regulated PTH secretion and clinical observations in affected subjects with activating CaR mutations.     

A novel calcium-sensing receptor antagonist transiently stimulates parathyroid hormone secretion in vivo

Circulating calcium (Ca(2+)) is a primary regulator of bone homeostasis through its action on PTH secretion. Extracellular Ca(2+) modulates PTH secretion through a cell surface G protein-coupled receptor, the calcium-sensing receptor (CaR). The expression of the CaR suggests a critical role in cellular regulation by calcium in various organs, including parathyroid gland, bone, and kidney. Despite an obvious pharmacological utility for CaR antagonists in the treatment of disease, only a limited number of such classes of compounds exist. We have identified a novel class of small molecules with specific activity at the CaR. This class of compounds is represented by compound 1. It possesses potent antagonist activity at the human CaR with IC(50) values of 64 nm and 230 nm in inhibiting intracellular Ca(2+) flux and inositol phosphate generation in vitro, respectively. When administered to male rats in vivo, compound 1 robustly increased serum PTH levels. The stimulation of PTH secretion was rapid and transient when administered either iv or orally. The pharmacokinetic profile of compound 1 after oral administration revealed that maximal plasma levels of compound were reached within 1 h and the half-life of the compound to be approximately 2 h in rats. These data describe a representative compound of a novel chemical class than previously described allosteric modulators that offer a new avenue for the development of improved treatments of osteoporosis.     

Magnesium promotes both parathyroid hormone secretion and adenosine 3',5'-monophosphate production in rat parathyroid tissues and reverses the inhibitory effects of calcium on adenylate cyclase

Reduced extracellular Ca2+ is known to promote PTH secretion, while severe Mg2+ depletion has the opposite effect. We have correlated the effects of Mg2+ and Ca2+ on parathyroid hormone (PTH) secretion and cAMP accumulation by rat parathyroid tissues in vitro with the effects of these two metals on adenylate cyclase activity in broken membrane preparations. PTH secretion was maximal at 0.5 mM Ca2+, falling to low levels as the Ca2+ concentration was increased to 2.5 mM. Deletion of Mg2+ from the medium resulted in a marked decrease in PTH secretion at any given Ca2+ concentration. At a constant Ca2+ concentration of 1 mM, both PTH secretion and cAMP production rose to maximal rates as the Mg2+ concentration was increased from 0 to 2 mM. The adenylate cyclase of rat parathyroid membranes was stimulated by both GTP and guanyl-5'-yl-imidodiphosphate [Gpp(NH)p]. EDTA-treated membranes could not be stimulated by Gpp(NH)p. Repletion with Mg2+ was more effective than repletion with Ca2+ in restoring responsiveness to the guanine nucleotide. When membranes were maximally preactivated by Gpp(NH)p and then assayed in the presence of variable concentrations of metal ions, enzyme activity was directly inhibited by Ca2+ and stimulated by Mg2+. Adenylate cyclase sensitivity to Ca2+ inhibition was dependent upon the Mg2+ concentration; in the presence of 0.6 mM Mg2+ a 50% inhibition was produced by 0.05 mM Ca2+, while in the presence of 8 mM Mg2+ a 10-fold higher Ca2+ concentration was required for a similar inhibitory effect. The results suggest that Ca2+ may decrease PTH secretion at least in part by a direct inhibition of adenylate cyclase. Mg2+ may promote PTH secretion either by enhancing the activation of adenylate cyclase by endogenous guanine nucleotides or by competing with Ca2+ for binding to a distinct regulatory site on the enzyme.     

Ca(2+) receptor from brain to gut: common stimulus, diverse actions.

An extracellular Ca(2+)-sensing receptor (CaR) plays central roles in Ca(2+) homeostasis by regulating parathyroid hormone (PTH)secretion and renal Ca(2+) handling. The CaR is also expressed in intestine and bone, where its functions in mineral metabolism are not yet well defined. The receptor is also present in various types of cells seemingly uninvolved in systemic mineral ion homeostasis (such as neuronal and glial cells in the brain and various epithelial cells), where its actions are poorly understood but might involve the regulation of local ionic homeostasis and/or diverse cellular processes, such as cellular differentiation and proliferation.     

Calcium-sensing receptor expression and signalling in human parathyroid adenomas and primary hyperplasia

OBJECTIVE: Both in vivo and in vitro evidence indicates that primary hyperparathyroidism is characterized by a reduced sensitivity to extracellular calcium ([Ca2+]o). The existence of alterations in the expression and signalling of calcium sensing receptor (CaSR) in parathyroid neoplasia is still uncertain. In order to clarify the role of CaSR in the reduced [Ca2+]o sensing of parathyroid neoplasia we investigated PTH secretion and intracellular effectors triggered by CaSR activation as well as the levels of expression of CaSR and CaSR coupled G proteins (Gq/G11) in parathyroid adenomas and primary hyperplasia. MATERIALS AND METHODS: The study included 27 parathyroid adenomas, 4 cases of primary hyperplasia and pools of normal parathyroid biopsies. Tissues were either snap frozen in liquid nitrogen or placed in sterile medium for cell dispersion. The effects of increasing [Ca2+]o on in vitro PTH release, intracellular cAMP levels and intracellular calcium ([Ca2+]i) in cells loaded with the Ca2 + indicator fura-2 were evaluated. CaSR mRNA levels were assessed by semiquantitative RT-PCR analysis, using GAPDH as internal standard, while CaSR protein was detected by western blot analysis using a specific polyclonal antibody. Purified antisera selective for G11alpha and Gqalpha were used to detect this class of proteins. RESULTS: In basal conditions (at 0.5 mM [Ca2+]o) in vitro PTH released ranged from 29.4 to 1186 pg/well/60 minutes. Increasing [Ca2+]o from 0.5 to 1, 2.5 and 5 mM caused a variable effect. One group (n = 7) showed a significant but partial reduction of PTH release (of 17 to 60% of basal levels) that occurred at physiological [Ca2+]o concentrations (1 mM) while the remainder showed either inhibition detectable only at 2.5 mM (n = 15) or total (n = 9) resistance to [Ca2+]o. In the responsive cells, [Ca2+]o (1-5 mM) caused a pertussis toxin-insensitive [Ca2+]i rise (ranging from 10% to 260%), due to Ca2+ release from intracellular stores, and an inhibition of forskolin-stimulated cAMP levels. By RT-PCR almost all tumours tested showed a substantial reduction in CaSR mRNA levels when compared to the normal tissue (CaSR/GAPDH ratio: 3.1 +/- 0.5 vs. 15.5 +/- 3.1; P < 0.001), which was confirmed by immunoblotting analysis demonstrating low levels of CaSR protein in tumour tissues. Moreover, low amounts of G11alpha and Gqalpha, the G proteins involved in CaSR coupling, were observed in the majority of pathological tissues. CONCLUSIONS: The study shows that the activation of the calcium sensing receptors expressed in adenomatous parathyroid glands modulates intracellular effectors in a similar way to those operating in the normal parathyroid. Although a reduction of calcium sensing receptor expression is probably involved in the poor inhibition of PTH release induced by [Ca2+]o, this is not the only factor altering [Ca2+]o sensing in parathyroid adenomas, since tumours characterized by different in vitro sensitivity to [Ca2+]o showed similar CaSR levels. The low content of G proteins of the Gq subfamily might represent an additional alteration leading to a defective [Ca2+]o sensing.     

Studies on the biosynthesis and secretion of parathyroid hormone in monolayer cultures of bovine parathyroid cells (I) (author's transl)]

We have developed a preparation of monolayer cultures of bovine parathyroid cells in order to elucidate the control mechanism of the biosynthesis and secretion of parathyroid hormone (PTH) at cellular level. Dispersion of parathyroid cells was performed by stirring minced bovine parathyroid tissues in Hanks' BSS containing 0.3 yields to 0.5 percent collagenase at 37 degrees C for 60 min. Dispersed cells were cultured at 37 degrees C in MEM-Hanks' BSS containing 10 percent fetal calf serum and 15 mM HEPES. On the 5th day of the culture, the medium was replaced with 1 percent BSA-MEM-Hanks-HEPES buffer, and the cells were incubated with 3H-leucine or in the media containing various concentrations of calcium, magnesium, PGE1, PGE2 or DBcAMP. At the end of incubation, the cells were detouched and homogenized in 8M urea, 0.2 N HCL and 0.01 M cysteine solution. The isolation of proparathyroid hormone (ProPTH) and PTH was performed through the preparation of TCA-powder followed by CMC column chromatography. PTH in the incubation medium was determined by radioimmunoassay. It was demonstrated that the monolayer cultures of bovine parathyroid cells were synthesizing ProPTH and converting it to PTH. The cultures exhibited linear secretion rates of PTH into the medium. The secretion of PTH was markedly increased by PGE1, PGE2 or DBcAMP in the range of 10(-7) yields to 10(-5)M in the former and 10(-5) yields to 10(-3)M in the latter, while calcium or magnesium changed secretion rate in the range of 0.3 yields to 4.4 mM.     

Pancreastatin, a presumed product of chromogranin-A (secretory protein-I) processing, inhibits secretion from porcine parathyroid cells in culture

Chromogranin-A, also referred to as secretory protein-I, is a 50K protein found in and secreted by endocrine cells, in which it is costored with the native hormone. Porcine chromogranin-A contains a sequence identical to pancreastatin, a 49-amino acid, C-terminally amidated peptide that has been isolated from porcine pancreas, suggesting that chromogranin-A is the precursor of pancreastatin. Pancreastatin has been found to be a potent inhibitor of glucose-stimulated insulin release. As it is possible that pancreastatin inhibits secretion from other chromogranin-A-containing tissues in which it may be formed, we tested its action on dispersed porcine parathyroid cells in culture. Secretion of chromogranin-A and PTH was up to 6-fold greater at 0.5 mM Ca2+ than at 3.0 mM Ca2+. Pancreastatin (1 nM) reduced the secretion of both chromogranin-A and PTH at 0.5 mM Ca2+ to approximately the levels found at 3.0 mM Ca2+, but did not affect secretion at 3.0 mM Ca2+. Pancreastatin (0.01-1.0 nM) inhibited secretion of chromogranin-A in a dose-dependent fashion. Preincubation of the cells with pancreastatin was not required for inhibition. Transfer of inhibited cells to medium without pancreastatin led to restoration of secretion within 90 min. Phorbol myristate acetate (1.6 microM) stimulated secretion of PTH and chromogranin-A at 3.0 mM Ca2+, but not at 0.5 mM Ca2+. Pancreastatin reversed this stimulation, demonstrating that its inhibition was independent of Ca2+ concentration. These results are consonant with pancreastatin playing a physiological role in modulation of secretion by the parathyroid and, by extension, other endocrine tissues.     

The effects of phorbol myristate acetate on the intracellular degradation of bovine parathyroid hormone

The suppression of PTH release by high extracellular calcium (Ca2+) has been associated with secretion of biologically inactive carboxyl-terminal fragments of PTH (C-PTH), while relatively more intact PTH is released under low extracellular Ca2+ conditions. In the presence of high extracellular Ca2+, phorbol myristate acetate (PMA) has been shown to stimulate PTH release to levels observed at low Ca2+, suggesting that protein kinase-C (PKC) is involved in the regulation of PTH secretion. We have examined the effect of PMA on PTH secretion and the release of PTH fragments at high and low calcium concentrations. Primary cultures of bovine parathyroid cells were incubated for 90 min in 0.5 mM (low) or 2.0 mM (high) Ca2+ with or without 1.6 microM PMA. Reverse phase HPLC using an 18-60% gradient of acetonitrile in 0.1% trifluoroacetic acid was performed on the medium from these incubations, and the eluant fractions were analyzed with a carboxyl (C)-terminal-specific PTH RIA. Medium from cultures exposed to low Ca2+ exhibited two large peaks of PTH immunoreactivity, coeluting with intact PTH-(1-84) and a synthetic human C-PTH-(39-84). PMA treatment at low Ca2+ resulted in the secretion of a greatly reduced amount of intact PTH, suggesting that PKC may increase the production of PTH fragment. At high extracellular Ca2+ PMA caused an increase in total immunoreactive PTH release similar to that seen at low Ca2+. However, on HPLC analysis, proportionally more PTH eluted in the position of the C-PTH fragment than was seen with low Ca2+ stimulation of PTH secretion. It, therefore, appears that the degradation of PTH to C-PTH may be linked to activation of PKC and can be separated from the Ca2+ regulation of PTH release occurring at the cell membrane.     

Calcium-regulated secretion of tissue plasminogen activator and parathyroid hormone from human parathyroid cells.

The effects of Ca and other agents on secretion of plasminogen activator (PA) and PTH have been examined and compared, using parathyroid cells obtained from the glands of chronic renal patients. During 2 weeks culture at different [Ca], the secretory rates of PA activity and PTH were parallel; steady-state secretion over 24-h periods was maximal at 0.5-0.9 mM Ca, minimal at 1.5-2.5 mM Ca, and the [Ca] at 50% suppression was 1.1 mM. At 2.5 mM Ca, two inhibitors of cellular proteolysis, 3-methyladenine and chloroquine, stimulated secretion of both PA activity and PTH. The results indicated that secretion of PA from human parathyroid cells is regulated similarly to that of PTH. The characteristics of human parathyroid PA were also examined using human parathyroid adenoma tissue. In homogenates, the highest specific activity of PA was in microsomal fractions. The Mr of PA from tissue and from culture media was 70 kilodalton by sodium dodecyl sulfate gel electrophoresis followed by zymography, or by Western blotting using antisera to human tissue PA (tPA). Enzyme activity was inhibited by incubation with antisera to tPA but not to urokinase. In contrast to bovine parathyroid cells that secrete a urokinase, human parathyroids apparently contain and secrete tPA.